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Electrochimica Acta
jou rn al hom epa ge: www.elsev ier .com/ locate /e lec tac ta
nhanced sensitivity for biosensors: Functionalized1,5-diaminonaphthalene-multiwall carbon nanotube composite film-modifiedlectrode
iali Zhang ∗, Shimei Yang, Hao Wang, Shaohui Wangepartment of Chemistry and Chemical Engineering, East China Jiaotong University, Nanchang 330013, PR China
r t i c l e i n f o
rticle history:eceived 22 June 2012eceived in revised form 22 August 2012ccepted 22 August 2012vailable online 30 August 2012
eywords:oly(1,5-diaminonaphthalene)
a b s t r a c t
A homogeneous electroactive poly(1,5-diaminonaphthalene) (P1,5DAN) and multiwalled carbon nano-tube (MWNT) composite film-modified electrode was fabricated by cyclic voltammetry and a castingmethod. The dispersion and morphology of the MWNTs/P1,5DAN composite film were investi-gated by scanning electron microscopy. The cyclic voltammograms of the electrode modified by theMWNTs/P15DAN composite film strongly depended on the film thickness and pH of the electrolyte solu-tion. Two absolutely isolated oxidation potentials were found as the MWCNTs were immobilized ontothe surface of P1,5DAN film in a pH 6.8 buffer solution containing ascorbic acid (AA) and uric acid (UA).
Both peak currents linearly increased with increased concentrations. The electrochemical behavior ofUA was not interrupted even in the presence of high-concentration AA given that AA had no observ-able electrochemical changes at the immobilized concentration. The electrocatalytic behavior of H2O2
was also investigated by steady-state amperometry for the immobilization of horseradish peroxidaseon the P1,5DAN film. The plot of the response current vs. H2O2 concentration was linear over the wideconcentration range of 0.015–5.37 mM.
. Introduction
Aromatic amine conducting polymers are mainly organic com-ounds that have extended single and double bonds alternating
n a �-orbital system. The electrons can be transferred alongolymer chains. The electrical properties of these polymers cane reversibly modulated by doping and undoping processes.oly(1,5-diaminonaphthalene) (P1,5DAN) synthesized by elec-rochemical oxidative polymerization has attracted considerablenterest because of its chelating and/or reduction properties [1].hese properties are the result of electron-donating groups (aminend secondary amino groups) on the polymer chains. P1,5DAN, as
functionality polymer, has been applied in electrocatalysts, asdsorbents, as well as it is used for chemical and biological sen-ors [2–4]. Incorporating different dopants into P1,5DAN film has
profound effect on the physical and chemical properties of theesulting material [5,6], such as the collection of heavy metal ions7–10] and coupling biomolecules with an extra free amine group
11,12]. In a previous research, P1,5DAN has been used to preparelectrochemical sensors for detecting cholesterol [11,12], hydro-en peroxide (H2O2) [13], domoic acid [14], and water [15]. The
sensors exhibit excellent permeability and selectivity. However,their poor sensitivity and the high compactness of the polymerfilm limit the diffusion of the substrates of various enzymatic reac-tions from solution to electrode surface. P1,5DAN reveals a verylow conductivity in a neutral solution, and excellent redox char-acteristics are only exhibited in acidic aqueous solutions at pH upto about 5 [16]. Therefore, fabricating a high-sensitivity P1,5DANfilm is a challenge. In particular, electron transfer in biosensorsystems is required to mediate between redox enzymes and con-ductive supports, a process that can be enhanced by increasing theelectrical conductivity of the electron-mediating system. Such anattempt has been made in the form of fabricating conducting poly-mer/carbon nanotube (CNT) composites [17,18]. CNTs serve as anexcellent matrix for promoting the electron transfer rate betweensolution and electrode in this composite. Polymers with CNTs canshift their electroactivity in a neutral pH environment [19–21]. Thepresence of CNTs in a polymer matrix could effectively improvesolubility without impairing physical properties [22,23]. Thus, thefabrication of conducting polymer/CNT composites that possessthe properties of the individual constituents is a great endeavor.The synergistic effect plays an important role in some applica-
tions [24,25]. A CNT/P1,5DAN composite doped by Au nanoparticlescan rapidly respond to fiber disaccharide dehydrogenated enzyme[26]. P1,5DAN, owing to its compatibility and conductivity, is usedas a mediator to facilitate the efficient binding of biomolecules
s well as shortening of the distance between the electrode sur-ace and the enzyme active site. In the current work, based on theifferent electrocatalytic activities of a modified electrode towardiomolecules, a multiwalled CNT (MWCNT)/P1,5DAN composite isrepared by assembling MWCNTs onto a P1,5DAN film. The aminond imine groups in the polymer chains are used for the simulta-eous determination of uric acid (UA) and ascorbic acid (AA). TheWCNTs/P1,5DAN film exhibits good selectivity, excellent peak
esolution, as well as efficient distinction in response to AA andA. The immobilization of horseradish peroxidase (HRP) on MWC-Ts/P1,5DAN allows for the low-potential detection and sensitiveetermination of H2O2, low detection limit, relatively wide linearesponse range, and a fast response time of 5 s.
. Experimental
.1. Reagents
1,5-Diaminonaphthalene (1,5-DAN; 99%) was purchased fromigma–Aldrich (USA). HRP (lot no. 100601, 250 units/mg) wasbtained from Shanghai Sanjie Co. (Shanghai, China). The MWCNTdiameter: 10–20 nm, length: 5–15 �m, purity >97%) was pur-hased from Shenzhen Nanotech. Port. Co. Ltd. (Shenzhen, China).A was from Xilong Co. (Guangdong, China). UA was frominepharm Chemical Reagent Co. Ltd. (Shanghai, China). All othereagents were analytical reagent grade and used as received. Allolutions were prepared with deionized water.
.2. Apparatuses
Electrochemical experiments were performed on a ModelHI430B time-resolved electrochemical quartz crystal microbal-nce (Chenhua, Shanghai). A conventional three-electrode systemas used, using glassy carbon (GC) electrode (GCE; diame-
er = 3 mm) as the working electrode, a platinum wire as theuxiliary electrode, and a Ag/AgCl (3 M KCl) electrode as theeference electrode. Electrochemical impedance studies were car-ied out on a Model CHI660C electrochemical station (Chenhua,hanghai). Scanning electron microscopy (SEM) measurementsere performed using a Zeiss-�IGMA (Germany) scanning electronicroscope. The samples were prepared by coating the MWCNT
uspension on indium tin oxide modified by P1,5-DAN.
.3. Preparation of solutions
The HRP (5 mg/mL) solution was prepared in phosphate buffer0.05 M, pH 6.8) and stored at 4 ◦C when not in use. The MWCNTs10 mg) were dispersed in 5 mL of N,N-dimethylformamide (DMF)nd ultrasonicated for 1 h to obtain a black suspension. This sus-ension was sonicated for 30 min before each film preparation.
stock solution of 0.1 M H2O2 was prepared in deionized water.his stock solution was further diluted to needed H2O2 solutiononcentrations.
.4. Fabrication of modified electrode
Preparation of the MWCNTs/P1,5DAN/GCEs (diameter = 3 mm)ere carefully polished with a 0.05 �m alumina slurry on a polish-
ng cloth, and ultrasonically washed in ethanol as well as water forbout 5 min, respectively. The GCE was activated in 0.1 M H2SO4sing cyclic voltammetry (CV) in the potential range of −0.3 Vo +1.5 V. [27] The P1,5DAN film was prepared in 0.1 M HClO4
olution containing 0.25 mM 1,5DAN by sweeping the potentialetween −0.6 and 1.2 V versus Ag/AgCl for 30 cycles at a scanate of 50 mV s−1. The P1,5DAN modified electrode was washedith deionized water to remove the physically adsorbed monomer
Acta 85 (2012) 467– 474
[23]. The immobilization of MWCNT onto the conducting polymer-modified electrodes was carried out as previously described [19].About 4 �L of MWCNT (2 mg/mL) suspension was dropped onto theP1,5DAN/GCE surface with a pipette. After the evaporation of DMFin air, the resulting MWCNTs/P1,5DAN/GCE was obtained.
2.5. Immobilization of HRP on the MWCNTs/P1,5DAN/GCE
About 4 �L of HRP (5 mg/mL) in phosphate buffer solution(PBS; 0.05 M, pH 6.8) was coated over the surface of MWC-NTs/P1,5DAN/GC and dried at ambient conditions. The modifiedelectrode was denoted as HRP/MWCNTs/P1,5DAN/GCE. Prior to theuse, the modified electrode was immersed in pH 6.8 PBS, which wasstirred for 30 min to remove the remaining adsorbed HRP. [28]
2.6. Electrochemical measurements
Electrochemical experiments were performed at room temper-ature using a model CHI430B instrument (Chenhua, Shanghai).The MWCNTs/P1,5DAN/GCE was used to determinate UA and AAsimultaneously using differential pulse voltammetry (DPV). H2O2content was estimated by the amperometric method at −0.15 V,and the solution was continuously stirred with a magnetic stirrer. Inall measurements, the solution was continuously purged nitrogento remove the air.
3. Results and discussion
3.1. Electroactivity of the MWCNTs/P1,5DAN composite film
The electroactivity of various electrodes, including bare GCE,P1,5DAN/GCE, MWCNTs/GCE, and MWCNTs/P1,5DAN/GCE, wereinvestigated by CV in 0.1 M PBS (pH 6.8). As shown in Fig. 1A,the electrode modified by the MWCNTs/P1,5DAN composite filmexhibits a higher electrochemical response (Fig. 1A-d) than thatsolely modified by P1,5DAN or MWCNT (Fig. 1A-b and 1A-c, respec-tively). The bare GCE only shows a weak response current in neutralsolution (Fig. 1A-a). The electrode modified by P1,5-DAN evidentlyimproves the electron transmission from electrode to solution. Theimmobilization of MWCNTs further accelerates the electrochem-ical response in neutral solution. The composite film is found tohave a high conductivity and an excellent conductive structure.The electrochemical response also depends on the thickness ofthe composite film. This thickness with a fixed MWNCT amountis controlled by changing the cycles of CV during the electrochem-ical polymerization process. The cycle number of polymerizationis selected within the range of 7–40. As shown in Fig. 1B, theelectrochemical activity of MWCNTs/P1,5DAN/GCE in 0.1 M PBS(pH 6.8) solution increases with the cycle number of polymeriza-tion within the range of 7–30. Subsequently, the current responserapidly decreases with increased cycles of polymerization. It isprobably ascribed to promote the electron transfer rate betweensolution and electrode in this composite due to an excellent matrixfor MWCNTs, while a thicker P1,5DAN film will lead to a weakersensitivity. This finding implies that the cooperation effect of MWC-NTs and P1,5DAN depends on the thickness of P1,5DAN. About 30cycles of polymerization favor the combination of MWCNTs andP1,5DAN owing to the good dispersing environment provided.
3.2. Morphology and electrochemical impedance spectra of thecomposite film
Fig. 1C shows the electrochemical impedance spectra of bareGCE, P1,5-DAN/GCE, and MWNCTs/P1,5-PAN/GCE, respectively,in K3Fe(CN)6/K4Fe(CN)6 solution. The interfacial electron-transfer
J. Zhang et al. / Electrochimica Acta 85 (2012) 467– 474 469
Fig. 1. (A) Electroactivities of the cyclic voltammograms of different electrodes in 0.1 M PBS (pH 6.8): (a) bare GCE, (b) P1,5-DAN/GCE, (c) MWCNs/GCE, and (d) MWCNTs/P1,5-DAN/GCE. (B) The peak current at the potential of −0.4 V of different cycle numbers of polymerization. (C) Electrochemical impedance spectra of bare GCE (a), P1,5-DAN/GCE(b), and MWCNTs/P1,5-DAN/GCE (c) in 0.5 M KCl solution containing 2.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1). (D) Enlarged impedance spectra of curve c. The frequency rangew
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as 1 Hz–10 kHz, and the amplitude was 5 mV.
esistance (Ret) of bare GCE was 296 � (curve a), which corre-ponds to the electron transfer limited process. However, Ret ofhe GCE modified by P1,5DAN dramatically increases (curve b).he electrochemical impedance spectra presents a bigger circu-ar arc, indicating that P1,5DAN film hinders the charge transfer.his result can be ascribed to the poor conductivity of P1,5DANlm in netural solution and the lower diffusivity of ions due tohe negative charge of ferri/ferro cyanide, which is adsorbed ontohe P1,5DAN film. Consequently, electrostatic repulsion occursetween the electrode surface and solution. Fig. 1D is the ampli-cation of curve c and d, which are the electrochemical impedancepectra of the MWCNTs/P1,5-DAN composite film and the immo-ilization of HRP, respectively. As shown in the inset of Fig. 1D,
inearity exists in the low frequency area after the immobilizationf MWCNT on the polymer film, and Ret decreases from 6000 � to8 �, and the Ret value only increases 7 � as the HRP was immobi-
ized onto the MWCNTs/P1,5-DAN composite film. The compositelm of Au nanoparticle (AuNP) and poly(1.8-diminonaphthalene)P1,8DAN) was reported to effectively bridge the electron com-
unication between the active center of HRP and electrode [13].n the interest of comparison between carbon nanotubes and Auanoparticle on the aspect, its Ret value (45 �) is lower than thatf the HRP/P1,8DAN/AuNP/GC (1625 �). This finding suggests thathe coordination of MWCNT reduces the adsorption of ferri/ferroyanide in P1,5DAN film, and promotes the diffusion of electroac-ive compounds from solution to the composite film. Hence, thentroduction of MWCNTs can largely improve the conductivity andtructure of P1,5DAN film.
The morphology of the MWCNTs/P1,5DAN film was charac-erized by SEM. As shown in Fig. 2A, the composite film is
homogeneously distributed modified electrode film withoutggregation. Fig. 2B is a magnified SEM image of the surface of
the composite film. The MWCNTs dispersed onto the surface ofP1,5DAN film have a uniform and highly entangled network struc-ture. This structure can be attributed to the hydrophilicity of theamino groups in the polymer chains, which are responsible forthe difficult dispersion of the MWNTs in the polymer matrix andthe relatively low solubility of the MWNTs in most solvents. Thecomposite film has loose and porous three-dimensional reticularstructures, which can not only largely increase the surface area ofthe composite film, but also improve the active combination withenzymes. This finding indicates that the composite film is propi-tious to the response of compounds and electron transmission dueto the cooperation between MWCNTs and P1,5DAN.
3.3. Electrochemical enhancement mechanism of the compositefilm
Compared with the electroactivity and electrochemicalimpedance spectra of different electrodes, a higher electrochem-ical response and a lower impedance resistance were found. Thecomposite film-modified electrode reveals excellent electroac-tivity. CNTs are extensively applied in electron devices due totheir excellent conductivity. In most applications, especially inelectrochemical sensors, CNTs are externally modified to avoidagglomeration, such as via carboxylation on the CNT surface. Inthe present work, MWCNTs can be homogeneously dispersed onP1,5DAN film without external modification (Fig. 2), and exhibitenhanced current response (Fig. 1A). This result indicates that thehydrophilicity of the amino groups in P1,5DAN chains plays a part
in the difficult dispersion of the MWNTs in the polymer matrix.The electrochemical enhancement mechanism may be consideredas the synergy effect between MWCNT and P1,5DAN. Electrontransfer easily occurs between electrode and film because the
470 J. Zhang et al. / Electrochimica Acta 85 (2012) 467– 474
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Fig. 2. SEM images of MWCNTs/P1,5-DAN film: morpholog
mino group of P1,5DAN induces a tight combination between theomposite film and GCE. The addition of MWCNTs also improveshe configuration of the film surface; its specific surface areaargely increases because of the three-dimensional reticulartructure. Therefore, the proton, which directly participates in theedox reaction, is easily enriched on the electrode surface. Numer-us absorbed protons result in decreased interfacial resistanceetween the solution and the composite film.
.4. CV response of AA and UA based on different electrodes
The separation of UA and AA in a solution is difficult using a con-entional solid electrode due to their similar oxidation potentials.ig. 3 shows the CV behavior of UA and AA at different electrodes.he CV behavior of bare GCE (Fig. 3a) presents an irreversiblerocess in a mixed solution, and only a broad oxidation poten-ial is observed at 0.37 V. The two potentials of UA and AA arendistinguishable. In the case of the P1,5DAN-modified electrode,ig. 3b shows two isolated peak potentials at 0.30 and −0.08 V,espectively, and a shift to the separate region compared with
he bare GCE. This finding can be ascribed to the structural differ-nce between UA and AA that results in the different selectivity of1,5DAN. However, the determination of UA and AA in their mixedolution is also difficult. The poor conductivity and compactness of
ig. 3. Cyclic voltammograms of bare GCE (a), P1,5-DAN-modified electrode (b),nd MWCNTs/P1,5-DAN-modified electrode (c) in a PBS (pH 6.8) solution containing0 �M UA and 2 mM AA. Scan rate = 100 mV s−1.
e composite film (A); MWNCT distribution on P1,5DAN (B).
P1,5DAN film affect the electroactive response in natural solution,indicating that the conductivity and surface structure of the mod-ified film play a key roles in electrochemical reactions. As shownin Fig. 3c, two absolutely isolated oxidation potentials at 0.008 and0.373 V are found upon the immobilization of MWCNTs onto thesurface of P1,5DAN film. The peak current significantly increasescompared with the electrode solely modified by P1,5DAN. The dif-ference between the oxidation peak potential is reached to 0.365 V,which is a sufficiently large magnitude to allow for the simulta-neous determination of AA and UA. It suggests that a synergy effectmay exist between MWCNTs and P1,5DAN, and this synergy caneffectively promote the catalytic oxidation reaction of UA and AA.Therefore, the MWCNTs/P1,5DAN modified electrode can be usedfor the simultaneous or selective determination of UA and AA.
3.5. Effect of pH
The sensitivity and transmission rate of the film to UA andAA depend on their charge states. These states are affected bythe solution pH because the oxidation of UA and AA involvesan acid deprotonation process. The correlation has been investi-gated within the pH range of 5.0–8.0 by CV. In the presence of ahigher proton concentration, the electrochemical response of UAand AA decreases with increased pH from 5.0 to 6.0 in Fig. 4A.Then, upon increasing pH from 6.0 to 8.0, the maximum peak cur-rent of UA and AA at pH 6.4 and 6.8, respectively, are observed.Their peak currents are lower than that at pH 5.0. It may be con-sidered as the coordination effect of the deprotonated P1,5-DANpolymer and the ionization of AA and UA. When the pH valuesare close to ionization equilibrium state of AA (pKa1 4.1) and UA(pKa 5.4), numerous adsorbed substances with negative charge areenriched on the electrode surface with a positive charge before pH6.0. Therefore, the response current will decrease as the pH valuesincrease until pH 6.0. However, In the process of pH values increase,ionization difficultly occurs for UA and AA at a conjugated alkalisolution (pH > pKa) [29], P1,5-DAN polymer accompanies with adeprotonated process as well. The response current of electrodewill increase with pH values increase until a maximum becausethe deprotonated P1,5-DAN is easy to combine the molecule of AAand UA. Thus, pH 6.4 is considered as the reasonable pH for detec-ting UA and AA. As shown in Fig. 4B, the UA and AA peak potentials
negatively shift with increased pH from 5.0 to 8.0. Their potentialchanges present a linear relationship with a correlation coefficientof 0.9973 with increased pH. This finding indicates that the electro-chemical redox is a proton-participating process, according to the
J. Zhang et al. / Electrochimica Acta 85 (2012) 467– 474 471
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ig. 4. Dependence of the peak current (A) and peak potential (B) for 10 �M UA
ifferent pH values. Scan rate = 100 mV s−1.
ernst equation (Ep = E0 + 0.059 m/n × pH, where m is the numberf protons and n is the number of electrons). The redox reactionechanism of UA and AA can be deduced from their linear slopes
f −57.2 and −35.6 mV/pH, respectively. The result reveals that theole ratios of the electrons and protons of UA and AA during the
edox process are 1:1 and 2:1, respectively.
.6. Behavior of UA and AA based on DPV
In contrast to CV, DPV has a higher current sensitivity and selec-ivity. Fig. 5 displays the behavior of UA and AA using the DPV
ethod. Two segregated oxidation peaks at the potentials of 0.39nd −0.02 V are obtained in a PBS (pH 6.4) solution containing UAnd AA. The difference is 0.41 V, indicating that the detection ofA is not hampered in the presence of AA, and both peak currents
inearly increase with increased concentration. Their changesresent a linear relationship with correlation coefficients of 0.9965nd 0.9976, respectively. The linear detection ranges of the con-entrations of UA and AA are 0.035–0.13 mM and 1.2–6.5 mM,
espectively. To confirm further this finding, a wider mole ratioange is investigated by changing the concentration of UA and fix-ng that of AA. Fig. 6 shows the dependence of UA detection onts concentration in the presence of 5.0 mM AA. With fixed AA
ig. 5. DPV curves of different UA and AA concentrations using MWC-Ts/P1,5DAN/GCE. UA concentration (�M): (a) 0, (b) 35, (c) 45, (d) 50, (e) 70, (f)0, (g) 110, and (h) 130. AA concentration (mM): (a) 0, (b)1.2, (c) 1.7, (d) 2.0, (e) 3.5,f) 4.5, (g) 5.5, and (h) 6.5.
mM AA on the pH using the MWCNTs/P1,5-DAN-modified electrode in PBS with
concentration, the UA concentration response range changesbetween 0.01 and 0.45 mM, the peak current change has a lin-ear relationship with a correlation coefficient of 0.9971, whereasthe peak current of AA does not obviously change. This finding ispossibly ascribed to the facile removal of the oxidation productof AA from the electrode. [30] For UA, low concentrations can bedetected until 10 �M. The potential differences between UA andAA are always maintained at 0.41 V, implying the determination ofUA and AA on the MWCNTs/P1,5DAN/GCE electrode is relativelyindependent. The result also illustrates that DPV is propitious tothe detection of UA and AA compared with CV.
3.7. Electrocatalytic reduction of H2O2 on theHRP/MWCNTs/P1,5DAN electrode
MWCNTs, which are modified by carboxylic groups, can stronglybond HRP on their surfaces. The immobilization of HRP on the sur-faces of different conventional electrode materials gives rise to awide variety of modified electrodes. These electrodes exhibit an
electrocatalytic activity depending on the nature of the polymermatrix. P1,5DAN can immobilize MWCNTs by the combination ofthe noncovalent bond �–�, and bond HRP through electrostaticattraction between protein and the protonated P1,5DAN without
Fig. 6. DPV curve using MWCNTs/P1,5-DAN/GCE in PBS (pH 6.4) containing 5 mMAA in the presence of different UA concentrations. UA concentration (mM): (a) 0.01,(b) 0.02, (c) 0.04, (d) 0.07, (e) 0.09, (f) 0.12, (g) 0. 15, (h) 0.17, (i) 0.20, (j) 0.22, (k)0.25, (l) 0.30, (m) 0.35, (n) 0.4, and (o) 0.45. The inset is the plot of the peak currentagainst UA concentration.
472 J. Zhang et al. / Electrochimica Acta 85 (2012) 467– 474
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ig. 7. Cyclic voltammograms of bare GCE (a), MWCNTs/P1,5-DAN/GCE (b), and HRcan rate = 100 mV s−1.
eed for other crosslinking reagents [31]. To verify the effect ofhe catalase immobilized on the MWCNTs/P1,5DAN complex film,lectrochemical experiments in the absence and presence of H2O2ere carried out. Fig. 7A shows the cyclic voltammograms of differ-
nt modified electrodes at a scan rate of 100 mV s−1 in 0.1 M PBS (pH.8) solution. As shown in Fig. 7, a weak current response for bareCE is seen in the potential range of 0.0 V to −0.5 V. However, the
esponse current is greatly improved when the GCE is modified byhe MWCNTs/P1,5DAN complex film. This result implies that theddition of CNTs improves the proton transfer between solutionnd electrode due to the greatly decreased amount of anion at thelectrode surface. In contrast, the peak current slightly decreaseshen HRP is assembled onto the MWCNTs/P1,5DAN complex film,
ut a new reduction peak appears at −0.21 V. This finding can prob-bly be ascribed to the reduction peak of HRP. [32] To evaluate thectivity of HRP immobilized on the MWCNTs/P1,5DAN complexlm, the cyclic voltammograms of the different electrodes in theresence of H2O2 are shown in Fig. 7B. A current response to H2O2t −0.21 V can be found using the HRP/MWCNTs/P1,5DAN/GCE.his peak current increases with increased H2O2 concentration. Thelectrocatalytic reduction of H2O2 using HRP/MWCNTs/P1,5DANomplex film GCE was also studied by steady-state amperome-
ry, which is one of the most widely employed techniques foriosensors. The steady-state current depends on the concentra-ion of H2O2. The constant potential of the HRP-modified electrodeas set at −0.15 V vs. AgCl after amperometric measurement. The
ig. 8. (A) Steady-state amperometric responses of HRP/MWCNTs/P1,5-DAN modified GChe concentration of H2O2. (B) Amperometric response of HRP/MWCNTs/P1,5-DAN/GCE toncentrations of H2O2. Applied potential = −0.15 V.
CNTs/P1,5-DAN/GCE (c) in 0.1 M PBS (pH 6.8) (A) and containing 0.25 mM H2O2(B).
catalytic reduction current was monitored while amounts of H2O2were added. The stepwise increase in H2O2 concentration in thebuffers causes the corresponding growth of the catalytic reductioncurrents. As shown in Fig. 8A, a well-defined response is observedas the addition of H2O2 in the concentration range from 0.05 to0.5 mM. The plot of the response current vs. the H2O2 concentra-tion is linear over the wide concentration range of 0.015–5.37 mM(R2 = 0.997). The sensor has a detection limit of 5.63 �M estimatedat a signal-to-noise ratio of 3. An extremely attractive feature of theHRP/MWCNTs/P1,5DAN-modified GCE is its fast response time (ca.5 s) when the response current reaches 96%, and its highly stableamperometric response toward H2O2. These results reveal that thecooperation effect between HRP and the modified film favors theelectron and proton transfer between the solution and electrode.
Compared with the reported work concerning polyaniline andMWCNTs, [33], the sensitivity of HRP/MWCNTs/P1,5DAN-modifiedGCE was found to be 112 �A �M−1, which was lower than thatof the HRP/MWCNTs/PANI-modified GCE (44.3 �A mM−1) proba-bly due to the low conductivity of P1,5DAN corresponding to PANI.However, it exhibits a broader concentration range from 0.015 mMto 5.37 mM than from 0.2 to 19 �M and faster response of 5 sthan 10 s for polyaniline matrix. Moreover, in the work, the reduc-
tion potential of −0.15 V for the amperometric determination ofH2O2 is lower than −0.1 V for the HRP/MWCNTs/PANI-modifiedGCE, where the risk for interfering reactions of other electroac-tive species in the solution was minimized and also where the
E to H2O2 in 0.1 M PBS (pH 6.8). The inset of A is the plot of the peak current againsto 0.05 M tyrosine, 0.05 M, glycine, 0.5 M glucose, 0.5 M UA, 0.5 M AA, and different
J. Zhang et al. / Electrochimica
Table 1Determination results of H2O2 in C1 and C3 samples.
ackground current and noise levels reached their lowest values.his indicates that the electrochemically active matrix is suitableor electrocatalytic reduction of H2O2.
.8. Stability, reproducibility and recovery
For H2O2 biosensors, long-term stability is one of the mostmportant properties. The stability of the HRP/MWCNTs/P1,5DANlm modified GCE was investigated by recording its cyclic voltam-ogram in a buffer solution (pH 6.8). When the biosensor was
tored in a refrigerator at 4 ◦C and measured every 7 days, no obvi-us change was found in the peak potentials. The enzyme electrodeetained 96% of its initial response to the reduction of 0.25 mM H2O2fter one week, and after two weeks, the reduction peak currentsecrease by less than 7%. This finding indicates that HRP moleculesan be firmly immobilized on the surface of the MWCNTs/P1,5DANomplex film. Therefore, the long-term stability can be attributedo the good biocompatibility of MWCNTs/P1,5DAN as well as thetrong interaction between the enzyme and MWCNTs/P1,5DAN. Tovaluate the practicability of the HRP/MWCNTs/P1,5DAN/GCE elec-rode, the electrode is used to determine the H2O2 concentrationn unknown concentration through the amperometric measure-
ent method. An unknown concentration sample (disinfector) wasbtained from the Hospital of East China Jiao Tong University,hich was diluted 10 times with pH 6.8 PBS. The sample (C3)as prepared by adding equivalent volume of an unknown con-
entration (C1) to a known concentration (C2). Table 1 shows theetermined values and the recovery values. Their analytical recov-ries were in the range 95–107%.
The repeatability of the current response of one enzyme elec-rode to 0.25 mM H2O2 was examined. The relative standardeviation (RSD) was 3.2% for 10 successive assays. The electrode-o-electrode reproducibility was estimated from the response to.25 mM H2O2 at 6 different enzyme electrodes. They yielded a.9% RSD. The good reproducibility may be ascribed to the firm
mmobilization between MWCNT and HRP and P1,5DAN throughhe combination of the noncovalent bond �–�, and bond HRPhrough electrostatic attraction between protein and the proton-ted P1,5DAN.
.9. Selectivity of the modified electrode
Selectivity is one of the important parameters for evaluatingiosensors. The effect of substances that may interfere with theesponse of the biosensor was studied. The selectivity of the mod-fied electrode was examined by amperometry at −0.15 V in theresence of 0.025 M H2O2 by adding different substances. Theiresponse currents are shown in Fig. 8B. The addition of two-foldoncentrations of glycine and tyrosine, as well as 20-fold concentra-ions of glucose and AA does not cause an observable interference.n the presence of 0.5 M UA, the response of the biosensor increases;ts ratio of the current of the coexistent solution (I(s+c)) to only H2O2I(s)) is 2.18. This result indicates that the interference of UA during2O2 examination cannot be prevented. Nevertheless, the response
urrent caused by UA is lower than that of H2O2. In fact, in theresence of UA and AA, the interfering mechanisms of UA and AAre similar during H2O2 detection, and the response of current ofA and AA will decrease with pH values increase since their pKa
[
[
[
Acta 85 (2012) 467– 474 473
values, while the response of UA is more sensitive than that of AAin a pH 6.8 solution [34].
4. Conclusions
A novel composite film was used to fabricate a third-generationbiosensor in this paper. MWCNTs and HRP have been successfullyimmobilized on the P1,5DAN film without using a cross-linkingagent. Two observable oxidation potentials, whose difference wasabout 0.37 V, were found in a solution where UA and AA coex-isted. In the case of fixed AA concentration, the peak current of UAchanged and exhibited a linear relationship with a correlation coef-ficient of 0.9981, whereas the peak current of AA had no obviouschange. Moreover, the direct reduction of H2O2 by the cataly-sis of immobilized HRP has been shown. Because of the netlikestructure of the composite film and high protein-binding capacitythrough electrostatic attraction between protein and the proton-ated P1,5DAN, HRP can be firmly immobilized on the composite filmmodified electrode. The HRP/MWCNTs/P1,5DAN-modified elec-trode exhibits good electrocatalytic activity, short response time,and long stability for H2O2 at −0.15 V. Therefore, The combinationMWCNT with P1,5-DAN, on one hand, improved the low conduc-tivity and the high compactness of the polymer film due to highspecific surface of MWCNT, on the other hand, shortened the dis-tance between the electrode surface and the enzyme active sitethrough the conjugated chain of P1,5DAN.
Acknowledgements
The project was supported by the National Natural ScienceFoundation of China (21164001) and the Natural Science Founda-tion of Jiangxi Province, China (2010GZH0029).
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